PROJECT SUMMARY Ewing sarcoma is a rare tumor that occurs in children and young adults. The driving oncogenic event in this disease is most commonly the translocation EWS/FLI. Similar translocations involving ETS transcription factors occur in other malignancies, including prostate cancer, broadening the potential relevance of our studies. Prior work has determined that EWS/FLI can lead to chromatin reprogramming by repressing some enhancers while activating others through direct binding. Our own data indicates that EWS/FLI also upregulates expression of lncRNAs. The primary goal of this proposal is to elucidate the molecular function of lncRNAs in Ewing sarcoma. Our preliminary data indicates that two lncRNAs, EWSAT1 and EWSAT2, may be involved in the oncogenic effects of Ewing sarcoma. Knock-down of either of these RNAs strongly decreases proliferation of Ewing tumors in vitro and in vivo. Furthermore, RNAseq analysis indicates that both of these lncRNAs repress a significant number of genes also known to be repressed by EWS/FLI. Our primary goal in this proposal is to elucidate the molecular function through which these lncRNAs act to promote Ewing sarcoma. We will focus our studies on determining how these lncRNAs lead to transcriptional repression. In Aim 1, we will elucidate define how expression of EWSAT1 and 2 alters key protein-protein interactions in Ewing cells. Specifically, we will test the hypothesis that EWSAT1 or 2 expression alters PPIs for EWS/FLI, EWS or hnRNPK. In Aim 2, we will test the hypothesis that EWSAT1 or 2 function by modulating the ability of EWS/FLI to bind to specific enhancers. ChIPseq and ChIRPseq will be used to determine how these lncRNAs interact with chromatin and how they alter binding of histones and other chromatin regulators. In Aim 3, we will elucidate the functional consequences of EWSAT1/2 loss or overexpression using Ewing patient derived xenografts and cell lines.